The frequency (frq) gene encodes central components of the transcription/translation-based negative-feedback loop comprising the core of the Neurospora circadian oscillator (FRQ). Posttranslational regulation associated with FRQ is surprisingly complex. Alternative use of translation initiation sites gives rise to two forms of FRQ whose levels peak 4-6 hr following the peak offrq transcript. Each form of FRQ is progressively phosphorylated over the course of the day, thus providing a number of temporally distinct FRQ products. The kinetics of these regulatory processes suggest a view of the clock where relatively rapid events involving translational regulation in the synthesis of FRQ and negative feedback of FRQon_frq transcript levels are followed by slower posttranslational regulation, ultimately driving theturnover of FRQ and reactivation of thefirq gene. The roles of mass spectrometry in this project are to (1) determine the molecular weight of FRQ expressed in different systems and tissues, (2) follow phosphorylation and locate modification sites, (3) document time-of-day changes, and (4) assess the nature of other posttranslational modifications, if any.